Project Summary Congenital aortic valve malformations, including bicuspid aortic valve, occur in approximately 2% of live births, and acquired heart valve diseases, such as calcific aortic valve disease, occur in about 2.5% of the U.S. population. It is theorized that mechanical forces influence both congenital and adult valve disease. Recently phenotypic and transcriptionally diverse aortic valve (AoV) cell populations were identified using single-cell RNA sequencing of maturing murine aortic and mitral valves. For the first time, three distinct valve endothelial cell (VEC) populations were identified in specific locations on the AoV. Each location experiences a unique combination of mechanical forces which could be important in diversifying VEC subpopulations. Importantly, one of these populations is present on the fibrosa side of the AoV where the VECs experience OSS. This population preferentially expresses the transcription factor Prox1, a master regulator of developmental processes, including cell fate and gene expression, in multiple organ systems. In the lymphatic system, Prox1 acts as a key regulator of lymphatic valve (LymphV) development and homeostasis in response to OSS. As Prox1 is present on the fibrosa side of murine embryonic day (E)13.5 semilunar valves and Prox1 remains present into adulthood, it is predicted that a similar mechanism is active during AoV development and homeostasis. The central hypothesis of this proposal is that Prox1 mediates aortic valve morphogenesis and homeostasis in response to changes in shear stress. Aim 1 will determine if fibrosa side-specific Prox1 promotes morphogenesis and homeostasis in the aortic valve by analyzing the expression pattern of Prox1 in the AoV of embryonic and adult mice throughout valve development and homeostasis. In parallel, the expression pattern of Gata2 and Cx37, Prox1?s inducer and downstream target in LymphV endothelial cells respectively, will also be analyzed (1A). Additionally, embryonic and adult endothelial-specific Prox1 loss-of-function mice will be utilized to study the effect Prox1 deletion has on Gata2 and Cx37 expression, as well as on valve morphology (1B). In Aim 2, the effects of altered shear stress on a Prox1 regulatory network and its relationship to valve homeostasis and disease will be analyzed. First, the expression pattern of Prox1, its lymphatic inducer (Gata2) and downstream target (Cx37) will be examined in porcine AoVs and in healthy and diseased human AoVs (2A). Next, the impact of physiologically relevant aberrant shear stress patterns on Prox1 in porcine AoV leaflets cultured ex vivo will be studied using a single cone-and-plate bioreactor (2B). Our long-term goal is to identify mechanosensitive mechanisms of AoV development, homeostasis and disease with potential therapeutic applications. This training plan combines studies of valve biology and bioengineering with translational implications. My career goal is to become a leader in understanding how the mechanical environment influences cardiovascular development, homeostasis and disease.